Posted 16 June 2009 - 04:42 AM
I have been PCR for a while now but this problem is really blocked me. I have trying to troubleshoot for a while now with no real success. First, I am trying to set up an accurate genotyping PCR to identify a specific transgene in a mouse. My first issue was that the transgene is quite homologeous to the endogenous gene and so, finding a primer pair that did not amplify the endogenous gene was difficult. The primer set that I have now, well, I am not sure quite what to think. I try repeating PCR reactions and I do not always get the same positive and negative. Than, sometimes, I also get a band in my water control. So, I changed all my reagents (including the water), cleaned my pipettes. I have now been trying to use different amount of DNA to see if that was the source of my variability. So, I repeat the same samples with 4 different volumes of DNA. I make 4 master mix in which I only vary the amount of water. The results are again somewhat variable and I occasionnally get 1 of the 4 water controls with a band! I thus repeated the experiment using the pipettes and tips from another lab but still, got the same thing. I am completely at a lost, does anybody have any suggestion?
Posted 16 June 2009 - 05:46 AM
Hope it`s not the case...
Posted 16 June 2009 - 07:25 AM
I may be able to help you out. First off, I will need you to send me all the information you have about your assay to this email: firstname.lastname@example.org. Before starting any discussion about what may be going on with your assay (contamination, etc), I will need to determine if your assay design makes sense ("I am not sure quite what to think" about your assay is not a good place to start when validating a genotyping assay). It is very easy to spend a ton of time running experiments that will never work when the assay itself is not detecting what it should. DNA sequences of your wildtype and transgene are critical for this.
In the mean time I suggest you go back and spend some more time on your assay design before you start ordering new primers, preparing new solutions, etc.
Posted 21 June 2009 - 04:37 PM
I dissolve my primers with water. Although that could have been a possibility, you would think that if something in my PCR mix is contaminated, then you would have bands in all the reactions that used that mix, right? But that is not the case. In addition, when I made 4 mixes simultaneously for different volumes of DNA, only 1 out of the 4 had a band in it but all of them had the some amount of primers, dNTP, buffer, taq and Mg, just different amounts of water! So, again, I would have thought that if I had a contamination in one of those reagents, I would get it in all my mixes, no?
Maybe your primer are contaminated? What did you use to dissolve them?
Hope it`s not the case...